Abnormal protein folding and aggregation of malfolded proteins likely contributes to neuronal death and dysfunction in chronic neurodegenerative disorders and in more acute brain insults such as stroke and trauma. Proteasomes, the complexes that perform the majority of cellular protein degradation, degrade these damaged and malfolded proteins. In addition, inhibition of proteasome function by such abnormal proteins might contribute to neuronal dysfunction in these disorders. Other components of the ubiquitin-proteasome pathway, most notably heat shock proteins, have been explored as therapeutic targets for neurodegenerative disorders and stroke, but proteasomes are molecularly complex structures and have been difficult to target pharmacologically or genetically. We have observed an enhancement of proteasome activity in cultured neurons treated for 20 hr with very low levels of proteasome inhibitors. We propose to use this novel method for enhancing neuronal proteasome activity to test the hypothesis that enhancement of proteasome activity reduces neuronal vulnerability to oxidative injury and ischemia. We hypothesize that the neuroprotective effects of proteasome inhibitor pretreatment are mediated by enhancement of proteasome activity, but will test alternative possibilities. We will quantify changes in expression of proteasome subunit proteins and mRNAs in proteasome inhibitor-pretreated neurons, and will test the ability of overexpression of specific proteasome subunits to enhance proteasome activity and attenuate neuronal injury. The goals of this project are to 1) demonstrate that proteasome inhibitor pretreatment enhances proteasome function, 2) to show that enhancement of proteasome activity is neuroprotective, and 3) to define molecular targets for therapeutic manipulation of proteasome activity.